Mass-Balanced Isotope Tags for Protein and Peptide Quantification using Mass Spectrometry

Abstract

DoctorAccurate identification and quantification of proteins expressed under different physiological conditions are very important in proteomics. Mass spectrometry (MS) combined with stable isotope-coded chemical tagging methods enables quantification and identification of protein. Recently, a number of the isobaric tags encoded by several stable isotopes have been introduced for simultaneous peptide sequencing and quantification in tandem MS (MS/MS). To expand the scope of isobaric tags in MS-based quantitative proteomics, we developed various H/D isotope-coded tags. Firstly, we present new isobaric tagging reagents called ？mass-balanced H/D-isotope dipeptide tags (MBITs)？ and methods for quantificaton. MBITs are N-acetyl dipeptides with amine-reactive O-succinimidyl ester (N-acetyl-Xxx-Ala-OSu, Xxx-tag). The H/D isotopes are incorporated into the N-acetyl group as CH3CO and CD3CO for LMBIT and HMBIT, respectively, while the mass balance is achieved by encoding alanine (Ala) with CD3 and CH3, respectively. To prove the concept, we use Ala in place of Xxx. The H/LAla-tag was prepared in either solution- or solid-phase. The coupling reaction between Ala-tag and a model peptide (angiotensin II) was optimized to achieve complete conjugation without side reactions. The isotopomeric model peptides differentially tagged by H/LAla-tag yield 2-plex isotope-coded fragment ions (LbS and HbS) by MS/MS. The measured ratios between LbS and HbS ([LbS]/[HbS]) reproduced well the premixed ratios of peptides. Secondly, MBITs are diversified as variable-mass tags by preparing seven more derivatives of Xxx-tags (Xxx = Ser, Val, Gln, His, Phe, Arg, and Tyr). Their performances in target conjugation, quantification linearity, and co-migration in liquid chromatography (LC) are checked with standard peptides (bradykinin and angiontensin II). H/LMBIT-linked isotopomeric peptides co-migrate in reverse-phase LC, and their tandem mass spectra exhibit the 2-plex signals as well as sequence ions in comparable abundances. The variation in Xxx changes the chromatographic elution profile as well as the ionization efficacy of target peptides. Of eight tags, His- and Gln-tags are best because of their strong and stable quantitation signals. His-tag shows the strongest signal due to histidine-specific fragmentation. Quantitation signal from Gln-tag show the least secondary fragmentation due to stable b-type ion. Diversified masses and properties of Xxx-tags provide the multiplexed protein quantificaton. Combination of (n？1) 2-plex Xxx-tags enables n-plex quantification. As a real application, four different Xxx-tags are applied to the multiplexed quantification of yeast heat shock protein (Hsp82p) expressed under five different chemical stresses in a single LC-MS experiment.Lastly, we provide a new strategy of protein quantification by presenting unique high-mass quantitation signals of MBITs attached to the lysine side chain. Although various isotope-coded isobaric tags have been widely used in MS-based quantitative proteomics, most of them provide quantitation signals in the low-mass region which is not accessible by common quadrupole ion trap tandem MS. Importantly, MBITs result in quantitation signals (L/HyS,K ions) in high-mass, noise-free region. Both arginine- and lysine-terminated tryptic peptides are examined by MALDI-TOF/TOF and ESI-QIT. Herein, we demonstrate for the first time the significance of high-mass signals originated from lysine-terminated peptides and present a lysine-tagging strategy of utilizing MBITs for MS-based quantitative proteomics using ion trap tandem MS